Methods of quantifying methotrexate metabolites

ABSTRACT

The present invention provides a method for efficiently converting methotrexate polyglutamates to methotrexate in a cellular extract by contacting the cellular extract with gamma glutamyl hydrolase under conditions suitable for efficient conversion of methotrexate polyglutamates to methotrexate.

BACKGROUND OF THE INVENTION

This invention relates generally to methods for monitoring drug therapyand, more specifically, to methods for monitoring efficacy and toxicityof anti-folate drug therapy.

Folate (folic acid) is a vitamin that is essential for thelife-sustaining processes of DNA synthesis, replication and repair.Folate is also important for protein biosynthesis, another process thatis central to cell viability. The pteridine compound, methotrexate(MTX), is structurally similar to folate and as a result can bind to theactive sites of a number of enzymes that normally use folate as acoenzyme for the biosynthesis of the purine and pyrimidine nucleotideprecursors of DNA and for the interconversion of amino acids duringprotein biosynthesis. Despite its structural similarity to folic acid,MTX cannot be used as a cofactor by enzymes that require folate, andinstead competes with the folate cofactor for enzyme binding sites,thereby inhibiting protein and DNA biosynthesis and, hence, celldivision.

The ability of methotrexate to inhibit cell division has been exploitedin the treatment of a number of diseases and conditions that arecharacterized by rapid or aberrant cell growth. As an example,autoimmune diseases are characterized by an inappropriate immuneresponse directed against normal autologous (self) tissues and aremediated by rapidly replicating T-cells or B-cells. Autoimmune diseasesthat have been treated with MTX include, for example, multiplesclerosis, rheumatoid arthritis, psoriasis, the autoimmune stage ofdiabetes mellitus (juvenile-onset or Type 1 diabetes), autoimmuneuveoretinitis, myasthenia gravis, autoimmune thyroiditis, and systemiclupus erythematosus.

Because many malignant cells proliferate more rapidly than normal cells,MTX can also be used to selectively impair cancerous cell growth. As aconsequence, methotrexate is a widely used anticancer agent, employed,for example, in the treatment of acute lymphocytic leukemia, breastcancer, epidermoid cancers of the head and neck, advanced mycosisfungoides, lung cancer, non-Hodgkins lymphomas, gestationalchoriocarcinoma, chorioadenoma destruens, and hydatidiform moles.

Despite its therapeutic efficacy for a wide variety of diseases andconditions, treatment with methotrexate can present a risk to thepatient. In particular, because MTX interferes with processes requiredfor replication and division of normal as well as diseased cells,inappropriately high levels of the drug can lead to destruction ofactively proliferating non-target tissues such as bone marrow andintestinal mucosa. MTX consequently is associated with renal and hepatictoxicity when administered in the “high-dose regimen” that is requiredfor some conditions. In addition, low-dose MTX therapy can lead totoxicity and unwanted side-effects in some patients, where the dosage isnot appropriate due to individual variability in pharmacokineticparameters influencing, for example, drug uptake, targeting andclearance. This situation is especially problematic in the treatment ofchronic conditions such as rheumatoid arthritis, where methotrexate canbe administered over a period of many years.

Because individual differences in pharmacokinetic parameters can bedifficult to predict, safe and effective methotrexate treatmentstrategies require that methotrexate or methotrexate metabolite levelsbe monitored in patients being treated. A variety of methods have beendeveloped for monitoring MTX drug concentrations in plasma includingbioassays, immunological detection and chromatographic assays. Suchplasma detection methods have been useful for monitoring high dose MTXtherapy in some clinical applications. However, these plasma detectionmethods have not been useful in monitoring low-dose methotrexatetherapy.

Methotrexate is metabolized upon uptake by mammalian cells, such thatone or more glutamyl moieties are added to MTX to yield a mixture ofmethotrexate polyglutamates (MTXPGs). The number of glutamyl moietiesthat can be added to MTX generally varies from two to seven. MTXPGs donot readily efflux from cells and thus are able to exert their cytotoxiceffects over long periods of time. Levels of intracellular MTXPGs havebeen shown to be higher in patients that responded to MTX therapy ascompared the intracellular levels in patients that did not respond.Currently available methods for measuring cellular MTXPG levels arebased on a dihydrofolate reductase enzyme assay in which MTXPG levelsare calculated based on inhibition of the dihydrofolate reductaseenzyme. However, the extent of enzyme inhibition in these assays isdependent upon the number of glutamyl residues attached to MTX,rendering an accurate determination of intracellular MTXPGs levelsimpossible by this method. The variability of dihydrofolate reductasebased assays can be further exacerbated in some situations becausefolates, which are present in different amounts depending upon anindividual's response to MTX therapy and the amount of folatecontributed by diet, also influence the results of the assay.

Thus, there exists a need for new methods for determining intracellularlevels of methotrexate polyglutamates and for monitoring the efficacyand toxicity of methotrexate therapy such as low-dose methotrexatetherapy. The present invention satisfies this need and provides relatedadvantages as well.

SUMMARY OF THE INVENTION

The present invention provides a method for efficiently convertingmethotrexate polyglutamates (MTXPGs) to methotrexate (MTX) in a cellularextract by contacting the cellular extract with gamma glutamyl hydrolaseunder conditions suitable for efficient conversion of MTXPGs to MTX. Themethod is useful for quantitating the level of MTXPGs in a cellularextract.

The invention further provides a method for determining a level ofmethotrexate polyglutamates in a cellular extract. The method includesthe steps of: (a) converting the MTXPGs to methotrexate in a cellularextract under conditions suitable for efficient conversion of MTXPGs toMTX, and (b) determining a level of the MTX, where the level of MTX canbe correlated with the level of MTXPGs in the cellular extract.

The invention also provides a method for determining a level ofmethotrexate in a cellular extract by (a) irradiating the cellularextract, thereby producing a fluorescent MTX photolytic product; and (b)determining a level of the fluorescent MTX photolytic product, whereinthe level of the fluorescent MTX photolytic product can be correlatedwith the level of MTX. By indirectly determining the intracellular levelof methotrexate polyglutamates, a method of the invention can be useful,for example, for monitoring the efficacy or toxicity of MTX therapy.

The invention provides a method of optimizing therapeutic efficacy orreducing toxicity associated with methotrexate therapy administered toan individual. The method includes the steps of: (a) convertingmethotrexate polyglutamates to MTX in a cellular extract from anindividual under conditions suitable for efficient conversion of MTXPGsto MTX; and (b) determining a level of MTX in the cellular extract, thelevel of MTX correlating with a level of MTXPGs, thereby determining alevel of MTXPGs in the cellular extract, wherein a drug or dosagesubsequently administered to the individual is selected based on thelevel of MTXPGs.

The invention further provides an isolated deglutamated compositionwhich is characterized as (a) present in a red blood cell extractfollowing deglutamation; (b) having a retention time of about 12 minutesin a mobile phase consisting of a 15-minute linear gradient from 2%acetonitrile/98% mobile phase A to 25% acetonitrile/75% mobile phase Aat a flow rate of 1 ml/min through a Terra MS 018 reverse phase column,where the reverse phase column has dimensions of 25 cm×4.6 mm and aparticle size of 5 micrometers and where the mobile phase A contains 10mM ammonium acetate, pH 6.5, and 0.06% hydrogen peroxide in water; and(c) having a fluorescence excitation spectrum with peaks at 299 nm and402 nm in water and a fluorescence emission spectra with a peak at 460nm in water.

The deglutamated endogenous composition of the invention can be producedby: (a) converting polyglutamated compounds in a red blood cellularextract to produce deglutamated RBC compounds; (b) fractionating thedeglutamated RBC compounds under conditions including passage of amobile phase consisting of a 15-minute linear gradient from 2%acetonitrile/98% mobile phase A to 25% acetonitrile/75% mobile phase Aat a flow rate of 1 ml/min through a Terra MS 018 reverse phase column,wherein the reverse phase column has dimensions of 25 cm×4.6 mm and aparticle size of 5 micrometers and wherein the mobile phase A includes10 mM ammonium acetate, pH 6.5 and 0.06% hydrogen peroxide in water; and(c) isolating a compound that elutes in a peak at 12 minutes, where thepeak is detectable by emission at 464 nm upon excitation with radiationat 400 nm following photolysis of the compound by irradiation at 254 nmfor 3 seconds in 80% mobile phase A/20% acetonitrile.

The deglutamated endogenous composition of the invention can combined,if desired, with a pharmaceutically acceptable carrier to produce apharmaceutical composition. Such a pharmaceutical composition can beuseful, for example, in a therapeutic method and can optionally includemethotrexate. According to the invention, the deglutamated endogenouscomposition of the invention is also provided as a kit for use in atherapeutic or diagnostic method.

A deglutamated endogenous composition of the invention can be useful forreducing toxicity associated with methotrexate therapy. A deglutamatedendogenous composition of the invention also can be useful in diagnosticmethods such as methods for determining an intracellular level ofmethotrexate or a method of optimizing therapeutic efficacy or reducingtoxicity associated with methotrexate therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structures of methotrexate and methotrexatepolyglutamates. (A) The chemical structure of methotrexate. (B) Thechemical structure for methotrexate polyglutamate, where n refers to thenumber of glutamates attached to methotrexate.

FIG. 2 shows the effect of mobile phase composition on formation offluorescent MTX photolytic products. (A) A plot of peak area detected atdifferent pH values for MTX photolytic product A or product B; (B) Aplot of peak area detected at different acetonitrile concentrations forMTX photolytic product A or product B; (C) A chromatogram of thephotolytic products of MTX; and (D) Fluorescence excitation and emissionspectra for photolytic product A of the compound eluting at 12.42 minfrom two separate chromatography runs.

FIG. 3 shows typical chromatograms of red blood cell extracts. (A) Atypical chromatogram of a deglutamated red blood cell extract followingphotolysis; (B) A typical chromatogram of a deglutamated,MTXPG₂₋₇-spiked red blood cell extract following photolysis; and (C)Fluorescence excitation spectrum of photolytic product A produced fromthe peak eluting at 12.5 minutes in the chromatogram of part B(indicated as “RBC”) overlayed with excitation spectrum for photolyticproduct A produced from a methotrexate standard in water (indicated as“water”).

FIG. 4 shows the conversion results obtained for various methotrexatepolyglutamates. (A) Timecourse of conversion of MTXPG₂ to MTX. (B)Timecourse of conversion of MTXPG₂₋₇ to MTX.

FIG. 5 shows a plot of detected MTX concentration versus theconcentration of MTXPG₂ or MTXPG₂₋₇ added to a red blood cell sampleprior to deglutamation. MTX from samples with MTXPG₂ is shown assquares. MTX from samples with MTXPG₂₋₇ is shown as triangles.

FIG. 6 shows characterization of a red blood cell patient sample. (A)Chromatogram of a deglutamated RBC extract from a patient havingreceived low-dose MTX therapy. (B) Fluorescence excitation spectrum ofphotolytic product A produced from the peak eluting at 12.42 minutes inthe chromatogram of part A (indicated as “RBC”) overlayed withexcitation spectrum for photolytic product A produced from amethotrexate standard in water (indicated as “water”).

FIG. 7 shows a fluorescence excitation spectrum obtained for the eluateat 12.02 minutes in the chromatograph of FIG. 6A.

DETAILED DESCRIPTION OF THE INVENTION

As disclosed herein, intracellular levels of methotrexate (MTX) werequantitated by fluorimetric detection of MTX photolytic products. Asdisclosed in Example I, a cell extract containing MTX waschromatographically separated and photolysed to yield MTX photolyticproducts which were of fluorimetrically detected. The method wasdemonstrated to detect MTX at low concentrations such as 5 nmol/L.

As further disclosed herein, methotrexate polyglutamates (MTXPGs) in acellular extract were efficiently converted to MTX by contacting theextract with plasma as a source of gamma glutamyl hydrolase. Efficientconversion of intracellular MTXPGs to MTX, combined with fluorimetricdetection of MTX photolytic products, allowed accurate quantitation ofcellular MTXPG levels as demonstrated in Example II. As furtherdisclosed in Example III, a method of the invention for quantitatingcellular MTXPG levels was useful in monitoring intracellular MTXPGlevels in patients undergoing low-dose MTX therapy. Thus, as disclosedherein, the methods of the invention can be useful, for example, fordetermining the percentage of MTX that has been metabolized to MTXPGs ina cellular extract. The methods of the invention also can be useful forquantitating cellular MTXPG levels in order to determine the efficacy ortoxicity of methotrexate therapy.

The present invention further is directed to the discovery of anendogenous compound that is present in deglutamated red blood cellextracts. As demonstrated in Example IV, the endogenous compound can bechromatographically separated from unrelated red blood cell components.The endogenous compound is characterized by (a) being present in redblood cells following deglutamation; (b) having a retention time ofabout 12 minutes in a mobile phase consisting of a 15-minute lineargradient from 2% acetonitrile/98% mobile phase A to 25% acetonitrile/75%mobile phase A at a flow rate of 1 ml/min through a Terra MS C18 reversephase column, which has dimensions of 25 cm×4.6 mm and a particle sizeof 5 micrometers, where mobile phase A contains 10 mM ammonium acetate,pH 6.5, and 0.06% hydrogen peroxide in water; and (c) having afluorescence excitation spectrum with peaks at about 299 nm and 402 nmin water and fluorescence emission at about 464 nm in water. Theendogenous compound can be, for example, photostable to UV light for atleast 3 seconds and further can be selectively present in a deglutamatedRBC extract but absent from an untreated RBC extract.

As further disclosed herein, detection or monitoring of the levels ofthe endogenous compound can be used to optimize therapeutic efficacy orreduce toxicity associated with methotrexate therapy. In particular, thelevels of MTX and the endogenous deglutamated compound can be monitoredand compared to optimize therapeutic efficacy or reduce toxicityassociated with methotrexate or other anti-folate therapy. Theendogenous compound also can function as an analog of folic acid and canbe a useful adjunct to methotrexate therapy. As an example, the isolatedendogenous compound can be administered to an individual undergoingmethotrexate therapy in order to reduce attendant toxicity.

Based on these findings, the present invention provides a method forefficiently converting MTXPGs to MTX in a cellular extract by contactingthe cellular extract with gamma glutamyl hydrolase under conditionssuitable for efficient conversion of MTXPGs to MTX. A method of theinvention can be useful, for example, for removing MTXPGs from acellular extract, for isolating MTX from a cellular extract or forquantitating the level of MTXPGs in a cellular extract as describedfurther below.

As used herein, the term “methotrexate” is synonymous with “MTX” andmeans a molecule having the structure shown in FIG. 1A. MTX includes, inpart, a 2,4-diamino substituted pterine ring moiety linked at the 6position to the amino group of a p-aminobenzoyl moiety, thep-aminobenzoyl moiety having a methylated amino group and being amidebonded to a glutamic acid moiety. Methotrexate functions as an inhibitorof dihydrofolate reductase (DHFR), decreasing the production oftetrahydrofolate (THF) from dihydrofolate (DHF). As a consequence, MTXindirectly inhibits purine and thymidine synthesis and amino acidinterconversion. MTX also exhibits anti-proliferative activity throughinhibition of thymidylate synthesis, which is required to synthesize DNA(Calvert, Semin. Oncol. 26:3-10 (1999)). MTX and its synthesis andproperties are described in further detail in U.S. Pat. Nos. 2,512,572;3,892,801; 3,989,703; 4,057,548; 4,067,867; 4,079,056; 4,080,325;4,136,101; 4,224,446; 4,306,064; 4,374,987; 4,421,913; and 4,767,859.Methods of using MTX to treat cancer are described, for example, in U.S.Pat. Nos. 4,106,488, 4,558,690, and 4,662,359.

Methotrexate, which is useful in the treatment of a variety ofautoimmune diseases and cancers, is typically administered by the oralor parenteral route. MTX is readily distributed to body tissues, whereit is transported into cells by a specific carrier system which includescomponents such as the reduced folate carrier, RCF1, and the folatereceptor. Due to its high polarity at physiological pH, MTX does notreadily pass through the cell membrane, and the majority of MTX enterscells via specific carriers. Once inside the cell, MTX is converted toMTXPGs by specific enzymes such as folylpoly-gamma-glutamate synthetase,which add one or more glutamic acid moieties, linked by iso-peptidicbonds to the y-carboxyl of MTX as described, for example, in Kamen,Semin. Oncol. S18:30-39 (1997).

As used herein, the term “methotrexate polyglutamate” is synonymous with“MTXPG” and means a derivative of MTX having two or more glutamateswhich are amide bonded to the p-aminobenzoyl moiety of MTX as shown inthe generalized structure of FIG. 1B. The number of glutamates in amethotrexate polyglutamate varies from two to seven or more; the numberof glutamate moieties can be denoted by “n” using the nomenclatureMTXPG_(n) such that, for example, MTXPG₂ is MTXPG having two glutamates,MTXPG₃ is MTXPG having three glutamates, MTXPG₄ is MTXPG having fourglutamates, MTXPG₅ is MTXPG having five glutamates, MTXPG₆ is MTXPGhaving six glutamates, MTXPG₇ is MTXPG having seven glutamates, andMTXPG₂₋₇ is a mixture containing MTXPG₂, MTXPG₃, MTXPG₄, MTXPG₅, MTXPG₅and MTXPG₇ with the ratio of the individual polyglutamated forms in themixture not defined. It is understood that, consistent with thisnomenclature, MTXPG₁ is synonymous with MTX.

An extract from cells that have taken up MTX will typically contain amixture of methotrexate polyglutamated species that differ in the numberof attached glutamate moieties. Such mixtures of MTXPGs can beefficiently converted to MTX by a method of the invention as set forthbelow and demonstrated in Examples II and III. A method of the inventioncan also be used to efficiently convert MTXPGs that are homogeneous withrespect to the number of attached glutamate moieties as demonstrated bythe efficient conversion of MTXPG₂ to MTX in Example II. In particularembodiments, the methods of the invention are used to efficientlyconvert an MTXPG_(n) to MTX, where n is any integer greater than 1.

In a method of the invention, MTXPGs can be converted to MTX by gammaglutamyl hydrolase. As used herein, the term “gamma glutamyl hydrolase”is synonymous with “GGH” and means a molecule that catalyzes cleavage ofa gamma glutamyl bond to remove a glutamate moiety from a polyglutamatedsubstrate. In one embodiment of the invention, a GGH catalyzes cleavageof a gamma glutamyl bond to remove a glutamate moiety from apolyglutamated substrate and lacks activity in cleaving the glutamatemoiety from a monoglutamated substrate. GGHs useful in the inventionincluding, but are not limited to, enzymes such as EC 3.4.19.9, or afunctional equivalent thereof including, for example, gamma-glu-Xcarboxypeptidase, folate conjugase, pteroyl-poly-alpha-glutamatehydrolase, carboxypeptidase G and lysosomal gamma-glutamylcarboxypeptidase. Examples of substrates of a gamma glutamyl hydrolaseare MTXPGs and other polyglutamated antifolates. It is understood that aGGH can catalyze removal of any number of glutamate residues that arelinked to the gamma carboxyl of a monoglutamated moiety of apolyglutamated substrate, and under certain conditions can yield amonoglutamated product. As an example, MTXPG_(n) includes n−1 glutamatesthat can be catalytically removed by GGH; MTXPG₂ includes a singleglutamate that can be catalytically removed by GGH; and MTXPG₇ includes6 glutamates that can be catalytically removed by GGH. As discussedfurther below, it is understood that the actual number of glutamateshydrolyzed, if any, depends on the conditions and length of time theenzyme is in contact with the polyglutamated substrate.

In one embodiment of the invention, a GGH is in contact with a cellularextract under conditions suitable for cleavage of a gamma glutamyl bondsuch that all but one glutamate moiety are cleaved from a polyglutamatedsubstrate. As an example, carboxypeptidase G is a GGH that underparticular conditions can convert MTXPG to MTX and, additionally, canremove the glutamate moiety from MTX to yield4-amino-4-deoxy-N¹⁰-methylpteroic acid (DAMPA). Carboxypeptidase G canbe used to convert MTXPG to MTX in a method of the invention underconditions that reduce or prevent the latter activity, such as in thepresence of an inhibitor, thereby removing all but one glutamate moietyfrom MTXPGs and converting the MTXPGs to MTX.

Native gamma-glutamyl hydrolase is a glycoprotein typically having amolecular mass of 80 to 120 kDa (Yao et al., Proc. Natl. Acad. Sci. USA93:10134-10138 (1996)). The protein portions of human and rat GGH areabout 35 kDa with multiple potential asparagine-linked glycosylationsites, rat GGH having seven and human GGH having four based on sequencehomology (Yao et al., supra, 1996). Depending upon the tissue, GGH isfound in lysosomal compartments or secreted (Yao et al., J. Biol. Chem.271:8525-8528 (1996)). GGH typically has an acidic pH optimum and isoften sulfhydryl- and Zn²⁺-dependent (Yao et al., J. Biol. Chem.271:8525-8528 (1996)).

A variety of GGHs are useful in the invention, including those withnaturally occurring and non-naturally occurring sequences, glycosylatedand non-glycosylated forms, species homologs, or in crude, partiallypurified or purified forms. GGHs useful in the invention include,without limitation, biochemically purified native proteins as well asrecombinant proteins and fragments or isoforms thereof that retain gammaglutamyl hydrolase activity. A variety of GGHs can be useful in theinvention including, but not limited to, mammalian, primate, human,non-human primate, bovine, porcine, rat, and murine GGHs. Furthermore,GGHs with improved or diminished enzymatic activity, stability or otherfunction as compared to naturally occurring (wild type) GGH can beuseful in the invention, as can point mutants, deletion or insertionmutants and fusion proteins including GGH or an active fragment thereof.In particular embodiments, the invention is practiced with a sourcecontaining a GGH with at least 40% amino acid identity to the human GGHprovided in Genbank accession NM_(—)003878, or with at least 50%, 60%,70%, 80%, 90% or more amino acid identity as compared to the human GGHprovided in Genbank accession NM_(—)003878.

A GGH useful in the invention can be provided, without limitation, asplasma or a fraction thereof; or as an extract from a cell expressingGGH or a fraction thereof; or in any purified or partially purifiedform. Under optimal conditions, a GGH can have, for example, an activityof at least 30% conversion of MTXPG₂ to MTX per hour, for example, atleast 25% conversion of MTXPG₂ to MTX per hour, or at least 10%conversion of MTXPG₂ to MTX per hour. A GGH can be a GGH of any speciesorigin, for example, a human GGH having NCBI Accession NM_(—)003878(see, also, Rhee et al., Mol. Pharmacol. 53: 1040-1046 (1998), and Yaoet al., Proc. Natl. Acad. Sci. USA 93:10134-10138 (1996)); the rat GGHhaving NCBI Accession NM_(—)012960 (see, also, Yao et al., J. Biol.Chem. 271: 8525-8528 (1996)); or the mouse GGH having NCBI AccessionNM_(—)010281 (see, also, Esaki et al., Gene 234:93-100 (1999)).

Plasma containing GGH can be used to efficiently convert MTXPGs to MTXas demonstrated in Examples II and III. As used herein, the term“plasma” means the acellular fluid portion of blood. Plasma useful in amethod of the invention can be obtained from any of a variety ofanimals. As non-limiting examples, plasma useful in the invention can behuman plasma, primate plasma, non-human primate plasma, rat plasma,mouse plasma or plasma from any other mammal. Plasma can be used in theinvention as fresh plasma or, alternatively, following storage underconditions that maintain GGH activity. Plasma can be obtained usingmethods well known in the art such as centrifugation of blood at lowspeed and isolation of the supernatant fraction. Plasma can also beobtained from a commercial source, such as Sigma (St Louis, USA), whichprovides lyophilized plasma (Cat. No. P9523). Exemplary conditions thatcan be used to store plasma useful in the invention include, withoutlimitation, lyophilization, refrigeration, freezing or a combinationthereof.

A cell expressing GGH also can be a source of GGH useful in a method ofthe invention. Such a cell can be any cell that expresses endogenous orrecombinant GGH or both. Many cancer cells, including, for example,human breast cancer cells, hepatoma cell lines and leukemia cell linesare known to express high levels of GGH and can therefore serve assources of GGH for use in the invention. See, for example, Rhee et al.,Cell Pharmacol. 2:289-292 (1995), and Pizzorno et al., Cancer Res.55:566-573 (1995).

It further is understood that GGH can be obtained from a recombinantcell that expresses exogenous GGH. Nucleic acid molecules encoding GGHare known in the art, as set forth above, or can be obtained by routinecloning methods including, for example, isolation of a GGH-encodingnucleic acid molecule from a cDNA library or genomic library with anatural or artificially designed gene-specific nucleic acid probe, orPCR amplification using a sequence specific primer. The GGH-encodingnucleic acid molecule can be cloned into an expression vector and usedto transform cells for recombinant expression of GGH by routine methods,as described for human and rat GHH, for example, in Rhee et al., supra,1998, and Yao et al., supra, 1996. Those skilled in the art will be ableto obtain recombinant cells expressing any of a variety of GGHs usingthese and other routine cloning methods that are well known in the artas described, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, 3rd ed., Cold Spring Harbor Press, Plainview, N.Y.(2001); and Ausubel et al. (Current Protocols in Molecular Biology(Supplement 47), John Wiley & Sons, New York (1999).

Purified or partially purified GGH also can be useful in a method of theinvention. As non-limiting examples, GGH can be obtained from a nativesource such as plasma or a tumor cell or a recombinant source such as abacterial, yeast or insect cell. GGH can be purified or partiallypurified using known methods as described, for example, in Scopes,Protein Purification: Principles and Practice, 3^(rd) Ed.,Springer-Verlag, New York (1994) and Coligan et al., Current Protocolsin Protein Science, John Wiley and Sons, Baltimore, Md. (2000).Isolation of GGH by these and other methods can be monitored using anantibody to GGH or an assay for GGH activity as described, for example,in Yao et al., supra, 1996, or Rhee et al., supra, 1998. As anon-limiting illustration, an E. coli cell lysate containing recombinanthuman gamma glutamyl hydrolase can obtained by sonicating E. coli cellsthat express the recombinant enzyme as described, for example, in Yao etal., supra, 1996. As a further illustration, recombinant human gammaglutamyl hydrolase can be expressed in intracellular or secreted formusing recombinant insect cells and purified with cation exchangechromatography or immunoaffinity chromatography as described, forexample, in Rhee et al., supra, 1998.

The methods of the invention involve contacting a cellular extract withgamma glutamyl hydrolase under conditions suitable for efficientconversion of MTXPGs to MTX. As used herein, the term “efficientconversion” means the production at least a 60% molar equivalent of MTXproduct compared to MTXPG substrate. Thus, the phrase “under conditionssuitable for efficient conversion of MTXPGs to MTX” means an environmentin which at least a 60% molar equivalent of MTX is produced from MTXPGs.In particular embodiments, the methods of the invention for efficientlyconverting MTXPGs to MTX produce at least 65%, 70%, 75%, 80%, 85%, 90%,95%, 98% or 99% molar equivalent of MTX product compared to MTXPGsubstrate. In addition embodiments, the methods of the invention produceat least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% molarequivalent of MTX product from MTXPG₂ substrate. Efficient conversion ofMTXPG₂ to MTX can occur within a time period of, for example, 2 hours, 4hours, 6 hours, 8 hours, 10 hours, 12 hours or longer. Thus, at least65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% molar equivalent of MTXproduct compared to MTXPG substrate can be produced, for example, within2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours or longer.

In one embodiment, a method of the invention is practiced by convertingMTXPGs to MTX under conditions that include a pH of at least 4. Themethods of the invention can be practiced, for example, under conditionsthat include a pH in the range of about 6 to 7. It is understood thatthe desired pH can be maintained using a buffer. Those skilled in theart can determine an appropriate buffer to maintain a particular pHbased on the pKa of the sample being used and known pKa values ofbuffers as described, for example, in Segel, Biochemical Calculations,2nd Ed., John Wiley and Sons, New York (1976). As illustrated in ExampleII, because plasma and RBCs are strongly buffered at physiological pH,addition of 100 mM phosphate buffer pH 4.5 to a mixture of plasma andRBC extract at a volume/volume ratio of 2 parts buffer:1 part RBCextract:2 parts plasma yields a final pH of about 6.5. Those skilled inthe art will recognize that buffers having a similar pKa can besubstituted for each other in order to maintain a desired pH in a methodof the invention. The concentration of phosphate in the conditions ofthe invention can be, for example, below about 400 mM such as below 350mM or below 300 mM.

By using an appropriate buffer, a method of the invention can bepracticed under conditions that include, for example, a pH of about 3.5,4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 or 8.5. In particularembodiments, a method of the invention is practiced under conditionsthat include a pH of at least 3.5, at least 4.0, at least 4.5, at least5.0, at least 5.5, at least 6.0, at least 6.5 or at least 7.0. In otherembodiments, a method of the invention is practiced with a pH in therange of 4.0 to 8.0, 4.5 to 8.0, 5.0 to 8.0, 5.5 to 8.0, 6.0 to 8.0, 6.5to 8.0, 7.0 to 8.0, 4.0 to 7.5, 4.0 to 7.0, 4.0 to 6.5, 4.0 to 6.0, 5.0to 7.5, 5.0 to 7.0, 5.0 to 6.5, 5.0 to 6.0, 6.0 to 7.0, or 6.5 to 7.0.

Conditions suitable for efficient conversion of MTXPGs to MTX caninclude, for example, a reducing agent as demonstrated in Example II,where 2-mercaptoethanol was included. The concentration of2-mercaptoethanol in a method of the invention can be, for example,about 50 mM or more. Other reducing agents can be useful in theinvention including, without limitation, dithiothreitol, dithionite,cysteamine or glutathione. It is understood that the concentration ofthe reducing agent can vary from those exemplified herein, so long asthe conditions lead to efficient conversion of MTXPGs to MTX.

Efficient conversion of at least 60% MTXPG₂₋₇ to MTX typically occurswithin about 3 hours or less using plasma as a source of GGH. As anexample, FIG. 4A shows that GGH provided in a plasma fraction convertedgreater than 60% MTXPG₂ to MTX in about 3 hours and greater than 90%MTXPG₂ to MTX in about 5 hours. As a further example, FIG. 4B shows thatGGH provided in a plasma fraction converted greater than 60% MTXPG₂₋₇ toMTX in about 2 hours and greater than 90% MTXPG₂₋₇ to MTX in about 5hours. Those skilled in the art will understand that the time for theconversion reaction can be altered from the times used for plasma GGH asexemplified herein depending, for example, on the use of a moreconcentrated or purified fraction of GGH, recombinant GGH or anothermolecule having GGH activity. As non-limiting examples, a method of theinvention can be practiced by contacting a cellular extract containingMTXPGs with plasma or another source of with gamma glutamyl hydrolasefor a time that is within the range of 2 to 15 hours, 2 to 10 hours, 2to 8 hours, 2 to 5 hours, 3 to 15 hours, 3 to 10 hours, 3 to 8 hours, 3to 5 hours, 5 to 15 hours, 5 to 10 hours, 5 to 8 hours, 6 to 24 hours, 9to 24 hours, 6 to 15 hours or 9 to 15 hours.

Plasma used as a source of GGH in a method of the invention can beprovided, for example, in a volume excess of about 2 fold or morecompared to the volume of a RBC extract, provided at a concentration ofabout 0.5×10⁹ red blood cells per 100 microliters. As exemplified inExample II, MTXPGs were efficiently converted to MTX in a RBC extractusing a 2 fold excess of plasma that had been reconstituted fromlyophilized form according to the manufacturer's instructions. Althoughmore concentrated conditions can be used in a method of the invention,precipitation of some of the MTXPGs by hemoglobin in the mixture canreduce the amount that is converted to MTX. Higher volume excess ofplasma to RBC extract can also be used such as 2.5 fold, 3 fold, 3.5fold, 4 fold or higher volume excess of plasma compared to RBC extract,so long as MTXPGs are efficiently converted to MTX. Those skilled in theart will recognize that dilution of RBC's in a method of the inventioncan be achieved in other ways including, without limitation, increasingthe volume of buffer present or decreasing the number of RBC's present.For example, when purified GGH or GGH from recombinant cells or enrichedplasma fractions is used in place of plasma in a method of theinvention, the concentration of the RBC's can be diluted, if desired,using buffer.

In a method of the invention MTXPGs are efficiently converted to MTX ina cellular extract. As used herein, the term “cellular extract” means amixture containing a heterogenous plurality of cellular components. Acellular extract useful in the invention can contain, for example, aheterogeneous plurality of soluble cellular compounds, proteins ormetabolites. Heterogeneity of a cellular extract can be characterized byvarious criteria. According to one criteria, a cellular extract usefulin the invention can be heterogeneous with respect to the variety ofcellular components present in the extract. In this regard, a cellularextract can contain, for example, at least 100, 1000, 1×10⁴ or 1×10⁵ ormore different cellular components, for example, at least 100, 1000,1×10⁴ or 1×10⁵ or more different proteins. Heterogeneity can also beexpressed as the percent of the total number of different components ofthe cell from which the extract is derived. For example, a cellularextract can contain cellular components representing at least 5%, 10%,15%, 20%, 25%, 50% or 75% of the variety of components present in thecell from which the extract was derived. Heterogeneity can also bedetermined based on the percentage of any one cellular component in acellular extract compared to the total amount of other components in thecellular extract. A cellular extract useful in the invention can have,for example, a cellular component that represents at most about 90%,80%, 70%, 60%, 50%, 25%, or 10% of the total amount of other cellularcomponents in the mixture. A cellular extract used in a method of theinvention can contain mixtures of components such as proteins,components that are larger than 100 Da or components that absorbradiation between about 303 nm and 313 nm or at about 370 nm.

A cellular extract that is useful in a method of the invention can beany cell extract that contains one or more MTXPGs. Additional exogenousMTXPGs can be added to a cellular extract as set forth in Example II.The addition of one or more exogenous MTXPGs into a cellular extract canbe useful for determining a standard curve for quantitating MTX or forotherwise testing or optimizing MTX detection conditions. A cellularextract containing MTXPGs can be obtained by adding MTX to a cellularextract and allowing polyglutamation to occur in vitro. Thus, a methodof the invention can be used to monitor or determine polyglutamationactivity of a cell or a component thereof, such asfolylpoly-gamma-glutamate synthetase. A cellular extract can also beprepared from a cell isolated from an individual that has beenadministered methotrexate by any route. Efficient conversion of MTXPGsto MTX in a cellular extract obtained from an individual undergoinglow-dose MTX therapy is disclosed in Example III. The method of theinvention for efficiently converting MTXPGs to MTX in a cellular extractfrom an individual treated with methotrexate can be useful foroptimizing therapeutic efficacy or reducing toxicity associated with MTXtherapy in the individual, as set forth in greater detail below.

The methods of the invention are well suited to efficiently convertingMTXPGs to MTX in a red blood cellular extract as demonstrated inExamples II and III. The conditions exemplified herein can also bereadily applied to other types of cellular extracts in order toefficiently convert MTXPGs to MTX. The cellular extract can be from acell that is a target for MTX therapy or otherwise is a cell indicativeof efficacy or toxicity of MTX therapy. Non-limiting examples ofcellular extracts that are useful in the invention include extractsprepared from tissue biopsies, erythrocyte extracts, neutrophil extractsand leukocyte extracts. Additional cellular extracts useful in theinvention include neoplastic or cancer cell extracts such as thoseobtained from any of the specific cancers set forth below. Cellularextracts useful in a method of the invention further include, withoutlimitation, mammalian cellular extracts, primate cellular extracts,human cellular extracts, non-human primate cellular extracts, ratcellular extracts, mouse cellular extracts, cat cellular extracts, dogcellular extracts, bird cellular extracts and horse cellular extracts.

A cellular extract useful in the invention can be obtained from anyindividual treated with methotrexate therapy, including low-dose andhigh-dose therapy. In one embodiment, a cellular extract useful in amethod of the invention is from an individual having an autoimmunedisease. As used herein, the term “autoimmune disease” means a diseaseresulting from an immune response against a self tissue or tissuecomponent and can include a self antibody response or cell-mediatedresponse. The term autoimmune disease, as used herein, encompassesorgan-specific autoimmune diseases, in which an autoimmune response isdirected against a single tissue, such as Crohn's disease and ulcerativecolitis, Type I diabetes mellitus, myasthenia gravis, vitiligo, Graves'disease, Hashimoto's disease, Addison's disease and autoimmunegastritis; and autoimmune hepatitis. The term autoimmune disease alsoencompasses non-organ specific autoimmune diseases, in which anautoimmune response is directed against a component present in severalor many organs throughout the body. Such autoimmune diseases include,for example, rheumatoid disease, systemic lupus erythematosus,progressive systemic sclerosis and variants, polymyositis anddermatomyositis. Additional autoimmune diseases include perniciousanemia, autoimmune gastritis, primary biliary cirrhosis, autoimmunethrombocytopenia, Sjögren's syndrome, multiple sclerosis and psoriasis.One skilled in the art will know that a number of the autoimmunediseases set forth above have been treated with MTX therapy or can betreated with MTX therapy. One skilled in the art recognizes that themethods of the invention can be used with a cellular extract obtainedfrom an individual having any of the above or another autoimmunedisease.

In another embodiment a cellular extract useful in a method of theinvention is obtained from an individual having arthritis. As usedherein, the term “arthritis” means an inflammatory condition thataffects joints. Arthritis can be infective, autoimmune or traumatic inorigin; the term arthritis includes, but is not limited to, acutearthritis, acute gouty arthritis, bacterial arthritis, chronicinflammatory arthritis, degenerative arthritis (osteoarthritis),infectious arthritis, juvenile arthritis, mycotic arthritis, neuropathicarthritis, polyarthritis, proliferative arthritis, psoriatic arthritis,juvenile rheumatoid arthritis, venereal arthritis, and viral arthritis.

In a further embodiment a cellular extract useful in a method of theinvention is obtained from an individual having rheumatoid arthritis.Rheumatoid arthritis is a chronic systemic disease primarily of thejoints, usually polyarticular, marked by inflammatory changes in thesynovial membranes and articular structures and by muscle atrophy andrarefaction of the bones. Methotrexate is widely used in the treatmentof rheumatoid arthritis, and one skilled in the art recognizes that themethods of the invention can be practiced with a cellular extract froman individual having rheumatoid arthritis or another form of arthritis.

In another embodiment, a method of the invention is practiced with acellular extract from an individual having cancer. As used herein, theterm “cancer” is intended to mean any member of a class of diseasescharacterized by the uncontrolled growth of aberrant cells. The termincludes all known cancers and neoplastic conditions, whethercharacterized as malignant, benign, soft tissue or solid tumor andcancers of all stages and grades including pre- and post-metastaticcancers. The term cancer encompasses, without limitation, leukemias suchas acute lymphocytic leukemia and acute myelocytic leukemia; lymphomas;choriocarcinomas; head and neck cancers; and osteogenic sarcomas, eachof which are widely treated with methotrexate. The term cancer furtherincludes, but is not limited to, digestive and gastrointestinal cancerssuch as anal cancer, bile duct cancer, gastrointestinal carcinoid tumorsand colon cancer; esophageal cancer, gallbladder cancer, liver cancer,pancreatic cancer, rectal cancer, appendix cancer, small intestinecancer and stomach (gastric) cancer; breast cancer; ovarian cancer; lungcancer; renal cancer; cancer of the central nervous system; and skincancer. In a particular embodiment, a method of the invention ispracticed with a cellular extract obtained from an individual havingleukemia.

Rheumatoid arthritis and a variety of other autoimmune disorders such aspsoriasis, systemic lupus erythematosus, and graft-versus-host diseaseare typically treated with low-dose MTX therapy, which is also used insome cancer treatment regimens. In one embodiment, a method of theinvention is practiced with a cellular extract from an individualundergoing low-dose MTX therapy. As used herein, the term “low-dose MTXtherapy” means administration of MTX to an individual at a dose that isless than 40 mg/m² of body surface per week. Typically, low-dose MTXtherapy is administered at a dose in the range of 2.5 to 40 mg/m² ofbody surface per week depending upon the condition being treated.Low-dose MTX therapy is typically administered orally because relativelylow doses of MTX are absorbed by the gastrointestinal tract.

The methods of the invention can also be used with a cellular extractfrom an individual undergoing high-dose MTX therapy, for example, fortreatment of cancer. As used herein, the term “high-dose MTX therapy”means administration of MTX to an individual at a dose that is at least40 mg/m² of body surface per day, for example, at least 100, 500, 1000,1500, 3000 mg/m² or 5000 mg/m² of body surface per day. One skilled inthe art understands that high-dose MTX therapy can be administered atdoses up to 5 g/m² of body surface per day or higher depending upon thecondition or disease being treated. One skilled in the art recognizesthat the doses of MTX typically used in high-dose MTX therapy can beadministered, for example, intravenously or orally and that suchhigh-dose MTX therapy generally requires a period of recovery, which caninclude leucovorin rescue or other forms of folate replacement.

It will be understood that the dosage ranges of MTX set forth above inthe definitions of high- and low-dose MTX therapy are generalized withrespect to treatment of a variety of diseases and that the range of MTXdose that is administered for one disease can differ from the rangeadministered for another. Accordingly, a dose of 40 mg/m² of bodysurface per day, although generally considered high-dose MTX therapy,may be considered by those skilled in the art of cancer therapy as arelatively low dose for treating cancer. Similarly, a dose of 30 mg/m²of body surface per day, although generally considered as low-dose MTXtherapy, may be considered by those skilled in the art of rheumatologyas a relatively high-dose for treating rheumatoid arthritis.

Cellular extracts useful in the invention can be prepared from a cell ortissue using methods well known in the art. Those skilled in the artwill know or be able to determine an appropriate method for obtainingsource cells based on their location and characteristics. As an example,red blood cells and other blood cells can be obtained by harvestingthrough intravenous routes. Cells can also be removed from tissues, suchas cancer tissues, using known biopsy methods including, for example,those utilizing an open surgical incision, biopsy needle or endoscope.Cells will be lysed by any of a variety of means depending, in part, onthe properties of the cell. As non-limiting examples, cells can be lysedby mechanical disruption with glass beads, a Dounce homogenizer, frenchpress, or sonication; enzymatic disruption with lysozyme or other enzymethat degrades the cell wall; osmotic disruption or a combination ofthese methods.

A cellular extract useful in a method of the invention can be afractionated extract, which can be enriched in MTX or MTXPGs as comparedto the total extract. As an example, an extract can be fractionated bycentrifugation to remove insoluble material such as membranes and largecellular structures. Fractionation to separate MTX, MTXPGs, or analogsthereof, from other cellular components can include, without limitation,centrifugation, protein precipitation, liquid-liquid extraction,solid-phase extraction, or chromatography such as reverse phasechromatography, ion pairing chromatography or ion exchangechromatography, as described, for example, in Rubino, J. Chromatog.764:217-254 (2001). Additional methods that can be used to obtain andfractionate cellular extracts are well known in the art, as described,for example, in Scopes, supra, 1994, and Coligan et al., supra, 2000.

The methods described above for efficiently converting MTXPGs to MTXalso can be used to efficiently deglutamate other glutamated andpolyglutamated substrates, including polyglutamated antifolates. Anumber of antifolates useful in treating diseases and conditionscharacterized by aberrant cell proliferation can be polyglutamated incells. Such antifolates are advantageous for therapeutic use becauseintracellular derivatization of an antifolate with polyglutamateprevents the derivatized antifolate from being extruded from the cell,in contrast to monoglutamated antifolates, which are generally extruded.Thus, a polyglutamated antifolate is maintained in a cell for longperiods of time and, consequently, has prolonged cytotoxic activitywithout requiring high levels of administration.

Thus, the invention provides a method for efficiently deglutamating apolyglutamated substrate in a cellular extract by contacting thecellular extract with gamma glutamyl hydrolase under conditions suitablefor efficiently converting the polyglutamated substrate to amonoglutamated substrate or an unglutamated substrate. The inventionalso provides a method for efficiently deglutamating a polyglutamatedantifolate in a cellular extract by contacting the cellular extract withgamma glutamyl hydrolase under conditions suitable for efficientconversion of the polyglutamated antifolate to an antifolate. Suchconditions allow gamma glutamyl hydrolase to remove glutamates frompolyglutamated antifolates by cleavage of one or more iso-peptide bonds.

As used herein, the term “antifolate” means a molecule having structuralsimilarity to folate and activity as a folate antagonist against one ormore folate-dependent enzymes. Polyglutamylatable antifolates areantifolates that can be polyglutamated in a cell by an enzyme such asfolylpoly-gamma-glutamate synthetase. Examples of polyglutamylatableantifolates include, without limitation, aminopterin, raltitrexed,lometrexol, multitargeted antifolate (MTA), AQA, MTX and analogsthereof. Aminopterin, for example, possesses a hydrogen instead of amethyl group at position N-10 compared to the structure of methotrexate.Raltitrexed is a selective inhibitor of thymidylate synthase asdescribed, for example, in Kamen, Semin. Oncol. S18:30-39 (1997).Lometrexol selectively inhibits glycinamide ribonucleotideformyltransferase, the first enzyme involved in the pathway of de novopurine synthesis as described, for example, in Calvert, supra, 1999. MTAis an inhibitor of multiple folate-dependent enzymes, such asdihydrofolate reductase, thymidylate synthase, and glycinamideribonucleotide formyltransferase (see, for example, Calvert, supra,1999).

One type of antifolate useful in the invention is a methotrexate analog.As used herein, the term “methotrexate analog” means a molecule havingstructural and functional similarity to methotrexate. Methotrexateanalogs are functionally characterized, in part, by their inhibitoryactivity against dihydrofolate reductase. A methotrexate analog usefulin the invention acts as a substrate for polyglutamation in a cell by anenzyme such as folylpoly-gamma-glutamate synthetase. MTX analogsinclude, but are not limited to, 4-amino derivatives with halogensubstitution on the para-aminobenzoic moiety, such asdichloromethotrexate (see, for example, Frei et al., Clin. Pharmacol.Therap. 6:160-71 (1965)); 7-methyl substituted MTX (see, for example,Rosowsky and Chen, J. Med. Chem. 17:1308-11 (1974)); 3′,5′-difluoro MTX,(see, for example, Tomcuf, J. Organic Chem, 26:3351 (1961)); 2′ and 3′monofluorinated derivatives of aminopterin (see, for example, Henkin andWashtien, J. Med. Chem. 26:1193-1196 (1983)); and7,8-dihydro-8-methyl-MTX (see, for example, Chaykovsky, J. Org. Chem.40:145-146 (1975)). The skilled person understands that the methods ofthe invention can be used to optimize or monitor efficacy or toxicityassociated with methotrexate analogs or other antifolates in the samemanner as disclosed herein for monitoring methotrexate therapy.

The invention further provides a method for determining a level ofmethotrexate polyglutamates in a cellular extract. The method includesthe steps of: (a) converting the MTXPGs to methotrexate in a cellularextract under conditions suitable for efficient conversion of MTXPGs toMTX, and (b) determining a level of the MTX, where the level of MTX canbe correlated with the level of MTXPGs in the cellular extract. Step (a)of the method can further include, for example, contacting the cellularextract with gamma glutamyl hydrolase under conditions suitable forefficient conversion of MTXPGs to MTX, for example, any of theconditions set forth above. The method is useful for monitoring theefficacy or toxicity of MTX therapy, as set forth in further detailbelow.

As discussed above, the invention provides conditions suitable forefficient conversion of MTXPGs to MTX. Thus, the level of MTX in a“converted” cellular extract can be correlated with the level of MTXPGsin the cellular extract after correction for any baseline level ofmethotrexate. The term “level,” when used in reference to a particularmolecule in a sample, means an amount or concentration of the particularmolecule in the sample. As an example, a level of MTX within a cellularextract is the amount or concentration of MTX in the cellular extract; alevel of MTXPGs in a cellular extract is the amount or concentration ofMTXPGs in the cellular extract. In view of the above, it is understoodthat a level can be an absolute level such as a molar concentration orweight or a relative level such as a percent or fraction compared to oneor more other molecules in a sample. Specifically excluded from thedefinition of “level,” as used herein, are estimates or measurementsbased on enzymatic activity, such as inhibition of DHFR or other enzymeactivity.

Subsequent to efficient conversion of methotrexate polyglutamates tomethotrexate, the methods of the invention can include a step ofdetermining the level of methotrexate. Quantitative and accuratedetection of MTX at low levels in cellular extracts can be achievedusing the methods set forth below.

The invention also provides a method for determining a level ofmethotrexate in a cellular extract by (a) irradiating the cellularextract, thereby producing a fluorescent MTX photolytic product; and (b)determining a level of the fluorescent MTX photolytic product, whereinthe level of the fluorescent MTX photolytic product can be correlatedwith the level of MTX. By indirectly determining the intracellular levelof methotrexate polyglutamates, a method of the invention can be usefulfor monitoring the efficacy or toxicity of MTX therapy.

In a method of the invention, MTX can be irradiated to produce aphotolytic product. As used herein, the term “photolytic product” meansa molecule that is produced by cleavage of bonds in a substrate that iselectronically excited by radiation. The process of producing aphotolytic product is referred to as photolysis. Photolysis of MTX toproduce a “MTX photolytic product” can be performed, for example, withUV light, which is a term understood in the art to include anywavelength in the range of about 200 to 400 nm. It further is understoodthat any light source which produces UV light can be useful in a methodof the invention including, for example, a lamp such as an arc lamp orquartz halogen lamp, or a laser. As demonstrated in Example I, afluorescent MTX photolytic product was produced by irradiating MTX witha low pressure mercury UV lamp which emits radiation in the range of 225to 275 nm and has a peak output at 254 nm. It is understood that MTX canbe selectively irradiated with a particular wavelength in the UV rangeby using an appropriate light source, optical filter or combination ofthese components in accordance with their known optical characteristics.

A sample, such as a cellular extract, that contains MTX, can beirradiated according to a method of the invention for the appropriateperiod of time to yield a fluorescent MTX photolytic product, where thelevel of the fluorescent MTX photolytic product can be correlated withthe level of MTX in the sample. In particular embodiments, a method ofthe invention is practiced by irradiating a cellular extract containingMTX for 0.5 to 60 seconds. Shorter periods of irradiation within thisrange can also be useful in a method of the invention including, forexample, irradiation for 0.5 to 15 seconds. In further embodiments, thecellular extract is irradiated for 0.1 to 100 seconds, 0.2 to 60seconds, 0.5 to 60 seconds, 0.5 to 45 seconds, 0.5 to 30 seconds, 0.5 to20 seconds, 0.5 to 15 seconds, 0.5 to 10 seconds, 1 to 20 seconds, 1 to10 seconds, 2 to 20 seconds or 2 to 10 seconds. In additionalembodiments, the cellular extract is irradiated for 0.5 to 6, 0.5 to 5,0.5 to 4, 1 to 6, 1 to 5, 1 to 4, or 2 to 4 seconds.

As demonstrated in Example I, irradiation for 3 seconds yieldedprimarily fluorescent MTX photolytic product A. As further demonstratedin Example I, irradiation for 24 seconds yielded primarily fluorescentMTX photolytic product B. In view of the above, it is understood thatthe time of irradiation can be varied to produce the desired fluorescentMTX photolytic product having characteristic properties as desired for aparticular application. One skilled in the art further understands thatthe methods of the invention can be practiced by detecting a singlefluorescent MTX photolytic product such as MTX photolytic product A orMTX photolytic product B. The MTX photolytic products A and B havedifferent properties including, for example, characteristic fluorescenceexcitation and emission peak maxima, and characteristic fluorescenceintensity levels depending upon the pH and amount of acetonitrilepresent during detection (see Example I).

Photolysis of MTX can be carried out in the presence of hydrogenperoxide (H₂O₂) or another peroxide. As a non-limiting example, whenhydrogen peroxide is added, the final concentration can be 0.03% orhigher. In particular embodiments, the final concentration of hydrogenperoxide is in the range of 0.05% to 1%, 0.2% to 1% or 0.3% to 0.6%.

A level of MTX in a cellular extract can be determined based on thelevel of a fluorescent MTX photolytic product. The term level, when usedin reference to a fluorescent MTX photolytic product, means an amount orconcentration of the fluorescent MTX photolytic product. The amount orconcentration of a fluorescent MTX photolytic product can be determined,for example, based on the intensity of fluorescence from the photolyticproduct.

As used herein, the term “fluorescence” means an emission of photons inthe ultraviolet (UV), visible (VIS) or infrared (IR) region of thespectrum in response to electronic excitation by radiation. The term“fluorescent,” when used in reference to a MTX photolytic product, meansa photolytic product that emits photons in the UV, VIS or IR region ofthe spectrum in response to electronic excitation by radiation. Thus, afluorescent MTX photolytic product is a photolytic product derived frommethotrexate that emits photons in the UV, VIS or IR region of thespectrum in response to electronic excitation by radiation. Afluorescent MTX photolytic product can be characterized, for example, asemitting photons at a quantum yield of at least 0.01 when excited byradiation in solution. In particular embodiments, a fluorescent MTXphotolytic product is characterized by a quantum yield of fluorescencethat is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or higherwhen excited by radiation in solution.

A fluorescent molecule, such as a fluorescent MTX photolytic product,also can be characterized with respect to maximum emission wavelength ormaximum excitation wavelength. In particular embodiments, a method ofthe invention involves determining the level of a fluorescent MTXphotolytic product having a maximum excitation wavelength in the IR,red, orange, yellow, green, blue, violet or UV region of the spectrum.In additional embodiments, a method of the invention is practiced bydetermining the level of a fluorescent MTX photolytic product having amaximum emission wavelength in the IR, red, orange, yellow, green, blue,violet or UV region of the spectrum.

Fluorescence can be detected in a method of the invention using any of avariety of excitation sources and emission detectors. Excitation of afluorescent MTX photolytic product can be achieved with an excitationsource such as a lamp or laser including, without limitation, any ofthose described above in regard to photolysis. Excitation at aparticular wavelength or in a particular wavelength range can beachieved in a method of the invention using, for example, a laser thatis tuned to the desired wavelength or a lamp having an output thatincludes the wavelength range. An appropriate optical filter can beplaced between the excitation source and fluorescent MTX photolyticproduct to further limit the range of wavelengths contacting thefluorescent MTX photolytic product if desired. As shown in FIG. 2D andset forth in Example I, fluorescent MTX photolytic product A has twoexcitation peaks in the range of 240 nm to 420 nm, including the peakfrom about 240 nm to 300 nm and the peak from about 360 nm to 410 nm. Inparticular embodiments of the invention, a fluorescent MTX photolyticproduct can be detected by excitation at a wavelength in the range ofabout 240 nm to 420 nm, about 240 nm to 300 nm or about 360 nm to 410nm. If desired, the methods of the invention can include excitation ator near the peak of 275 nm or in a range near this peak including, forexample, excitation at a wavelength in the range of 250 nm to 290 nm or260 nm to 280 nm. An excitation at or near the peak of 385 nm or in arange near this peak can also be used including, for example, excitationat a wavelength in the range of 250 nm to 290 nm or 260 nm to 280 nm.

Emission can be detected from a fluorescent MTX photolytic product usinga detector such as, without limitation, a photomultiplier tube, diode,diode array or charge coupled device camera. A detector that detectslight at a particular wavelength or in a particular wavelength range canbe used in a method of the invention. If desired, an optical filter canbe placed between the fluorescent MTX photolytic product and thedetector to limit the range of wavelengths detected. As shown in FIG. 2Dand set forth in Example I, fluorescent MTX photolytic product A emitsfrom about 320 nm to 550 nm and has a primary peak from about 440 nm to520 nm. In particular embodiments of the invention, emission from afluorescent MTX photolytic product can be detected at a wavelength inthe range of about 320 nm to 550 or about 440 nm to 520 nm. If desired,the methods of the invention can include detection of emission at ornear the peak of 464 nm or in a range near this peak including, forexample, emission at a wavelength in the range of 450 nm to 510 nm or460 nm to 490 nm.

A level of MTX also can be determined using, for example, chromatographyor mass spectrometric analysis. MTX can be chromatographically separatedfrom other cellular components using, for example, reverse phasechromatography as set forth in Examples I through III and subsequentlyquantitated, for example, by comparison to known reference standards. Asdemonstrated herein, MTX was accurately detected using reverse phasechromatography on a high performance liquid chromatography system. As anon-limiting example, chromatographic analysis can include passing anMTX containing sample through a C18 reverse phase column in a mobilephase consisting of a linear gradient from 2% acetonitrile/98% mobilephase A to 25% acetonitrile/75% mobile phase A, wherein mobile phase Ais 10 mM ammonium acetate, pH 6.5.

The content of a solution that is used to detect MTX, or a photolyticproduct thereof, can be varied, for example, with respect to pH oracetonitrile content. The pH at which MTX, or a photolytic productthereof, is detected in a method of the invention can be in the rangeof, for example, about pH 2 to 8 or in the range of about pH 4 to 7. Inparticular embodiments, MTX, or a photolytic product thereof, can bedetected, for example, at pH 4, 4.5, 5, 5.5, 6, 6.5 or 7. The amount ofacetonitrile present during detection of MTX, or a photolytic productthereof, can be in the range of, for example, about 0% to 20% or about10% to 20%. In particular embodiments the amount of acetonitrile presentcan be, for example, 5%, 10%, 15% or 20%.

A reverse phase column useful for separating MTX from other cellularcomponents can have, for example, dimensions of 25 cm×4.6 mm, asexemplified herein. Columns having larger or smaller diameters, lengthsor both can also be used, for example, to accommodate larger or smallersample size. Flow rates can vary, for example, from 0.2 to 2.5 ml/min.As demonstrated herein, the flow rate for the mobile phase can be, forexample, 1 ml/min. However, the flow rate of the mobile phase can bealtered as desired. A slower flow rate, such as 0.8 ml/min, 0.5 ml/minor 0.2 ml/min, can be used, for example, with a smaller column or toincrease MTX retention times. Alternatively a faster flow rate, such as1.5 ml/min or 2.0 ml/min, can be used, for example, with a larger columnor to decrease MTX retention times.

A level of MTX in a sample also can be detected in a method of theinvention based on other properties of MTX including, for example,ultraviolet or visible light absorption properties, fluorescence,electrochemical properties, or mass. Methods for detection of MTX basedon these properties are known in the art as described, for example, inRubino, supra, 2001. As non-limiting examples, a level of MTXPG in asample can be determined by efficiently converting MTXPGs to MTX underconditions suitable for efficient conversion and detecting the resultingMTX with UV/Vis absorption spectroscopy, fluorimetry, electrochemicaldetection, or mass spectrometry. Those skilled in the art will know orbe able to determine an appropriate means for detecting methotrexatebased on the accuracy and sensitivity desired and the presence ofpotentially interfering substances in the particular sample beinganalyzed.

The invention provides a method of optimizing therapeutic efficacy orreducing toxicity associated with methotrexate therapy administered toan individual. The method includes the steps of: (a) convertingmethotrexate polyglutamates to MTX in a cellular extract from anindividual under conditions suitable for efficient conversion of MTXPGsto MTX; and (b) determining a level of MTX in the cellular extract, thelevel of MTX correlating with a level of MTXPGs, thereby determining alevel of MTXPGs in the cellular extract, wherein a drug or dosagesubsequently administered to the individual is selected based on thelevel of MTXPGs.

Intracellular levels of methotrexate polyglutamates can be associatedwith efficacy, with higher levels associated with toxicity. Bydetermining the intracellular level of methotrexate polyglutamates, themethods of the invention can be useful for adjusting the amount orfrequency of methotrexate therapy in order that the methotrexatepolyglutamates are within the therapeutic range and do not exceed thisrange, thereby producing undesirable toxic side effects. Any of avariety of types of cellular extracts can be useful in a method of theinvention for optimizing therapeutic efficacy or reducing toxicityassociated with methotrexate therapy. Cell extracts can be prepared fromany cell or tissue that is indicative of the efficacy or toxicity ofmethotrexate therapy such as a diseased cell or tissue, a target cellfor MTX therapy or a cell that is representative of the amount of drugin diseased cells. As used herein, the term “target cell for MTXtherapy” means a cell for which uptake of MTX is desired to treat adisease or condition. As non-limiting examples, cell extracts can beprepared from red blood cells, leukocytes, neutrophils, cancer cells,and tissue biopsies.

A method of the invention for optimizing therapeutic efficacy orreducing toxicity associated with methotrexate therapy can furtherinclude altering the methotrexate dosage subsequently administered tothe individual based on the determined level of methotrexatepolyglutamates. Where the determined level of MTXPGs is below atherapeutic range, the dose or frequency of methotrexate can beincreased. Where the determined level of MTXPGs is above a therapeuticrange, the dose or frequency of methotrexate can be reduced to avoidtoxicity. A therapeutic range can be determined from dose-responseinformation for a particular disease or condition and, if desired, forthe age, gender and medical condition of the individual being treated.As an example, a level of MTXPG determined in a red blood cellularextract can be compared to dose-response information correlating redblood cell methotrexate polyglutamate levels with reduction of anarthritis symptoms. Dose-response information also can be obtained usingwell known clinical procedures relevant to the particular pathologicalcondition.

In a particular embodiment of the invention, a cellular extract usefulin a method of the invention for optimizing therapeutic efficacy orreducing toxicity associated with MTX therapy can be obtained from anon-target cell for MTX therapy. As used herein, the term “non-targetcell for MTX therapy” means a cell for which uptake of MTX is notdesired. Such a non-target cell can be a normal cell that is sensitiveto levels of MTX or that is disposed to take up MTX that is administeredby a particular route. Determining a MTXPG level in a cellular extractfrom a non-target cell also can be useful to determine toxicity of MTXtherapy.

Methotrexate therapy can cause a variety of unwanted adverse effectsthat mimic folate deficiency including, for example, gastrointestinalintolerance, stomatitis, alopecia and cytopenia. Many adverse effects ofmethotrexate therapy are dose dependent and can be alleviated byadministration of compensatory doses of folate. Accordingly, a method ofthe invention for optimizing therapeutic efficacy or reducing toxicityassociated with methotrexate therapy can further include, if desired,altering the dose of folate, or a derivative thereof, subsequentlyadministered to the individual based on the determined level ofintracellular methotrexate polyglutamates.

The invention further provides an isolated deglutamated compositionwhich is characterized as (a) present in a red blood cell extractfollowing deglutamation; (b) having a retention time of about 12 minutesin a mobile phase consisting of a 15-minute linear gradient from 2%acetonitrile/98% mobile phase A to 25% acetonitrile/75% mobile phase Aat a flow rate of 1 ml/min through a Terra MS 018 reverse phase column,which has dimensions of 25 cm×4.6 mm and a particle size of 5micrometers, and where the mobile phase A contains 10 mM ammoniumacetate, pH 6.5, and 0.06% hydrogen peroxide in water; and (c) having afluorescence excitation spectrum with peaks at 299 nm and 402 nm inwater and a fluorescence emission spectra with a peak at 460 nm inwater. The endogenous compound can be further characterized as beingphotostable to irradiation with a 254 nm low pressure mercuryultraviolet lamp for 3 seconds, wherein following irradiation with 254nm for 3 seconds the fluorescence excitation spectrum of the isolateddeglutamated composition has peaks at 299 nm and 402 nm in water and theisolated deglutamated composition emits fluorescence at 464 nm in water.The endogenous compound can further be characterized, for example, ashaving a fluorescence emission peak at 460 nm in water.

The present invention further provides an isolated composition producedby: (a) converting polyglutamated compounds in a red blood cellularextract to produce deglutamated RBC compounds; (b) fractionating thedeglutamated RBC compounds under conditions comprising passage of amobile phase consisting of a 15-minute linear gradient from 2%acetonitrile/98% mobile phase A to 25% acetonitrile/75% mobile phase Aat a flow rate of 1 ml/min through a Terra MS 018 reverse phase column,which has dimensions of 25 cm×4.6 mm and a particle size of 5micrometers, and where the mobile phase A includes 10 mM ammoniumacetate, pH 6.5 and 0.06% hydrogen peroxide in water; and (c) isolatinga compound that elutes in a peak at 12 minutes, where the peak isdetectable by emission at 464 nm upon excitation with radiation at 400nm.

The invention also provides an isolated deglutamated composition, suchas the endogenous compound described in Example IV, together with apharmaceutically acceptable carrier. An isolated deglutamatedcomposition of the invention can be, for example, a folate derivative;in this case, a pharmaceutical composition containing the derivative canbe useful as a surrogate for folate in any of a variety of applications.Such a pharmaceutical composition can be useful, for example, in amethod of reducing toxicity associated with methotrexate therapy. Thus,the present invention also provides a method of reducing toxicityassociated with methotrexate therapy by administering to an individualtreated with methotrexate therapy an effective dose of a pharmaceuticalcomposition of the invention.

Any of a variety of pharmaceutical compositions are encompassed by theinvention. As one example, a deglutamated composition can beadministered as a solution or suspension together with apharmaceutically acceptable carrier. Such a pharmaceutically acceptablecarrier can be, for example, water, sodium phosphate buffer, phosphatebuffered saline, normal saline or Ringer's solution or otherphysiologically buffered saline, or other solvent or vehicle such as aglycol, glycerol, an oil such as olive oil or an injectable organicester. Pharmaceutically acceptable carriers can be sterile orsubstantially free from contaminating particles and organisms. Acomposition of the invention that includes a pharmaceutically acceptablecarrier can be of sufficient purity and quality for use in humans. Apharmaceutical composition of the invention containing a deglutamatedcompound can optionally include methotrexate.

A pharmaceutical composition of the invention can be formulated forparenteral administration such as subcutaneous, intraperitoneal,intramuscular, intravenous, intradermal, intracranial, intratracheal,and epidural administration. A pharmaceutical composition of theinvention also can be formulated for oral, topical (including buccal andsublingual), rectal, ophthalmic, nasal, intrauterine or vaginaladministration. A pharmaceutical composition of the invention can bepresented in unit dosage form and can be prepared by pharmaceuticaltechniques well known to those skilled in the art. Such techniquesinclude the step of bringing into association the active ingredient anda pharmaceutical carrier or excipient.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injectable solutions including any of thepharmaceutically acceptable carriers described above. The solutions canadditionally contain, for example, one or more anti-oxidants, buffers,bacteriostats or solutes which render the formulation isotonic with theblood of the intended recipient. Other formulations include, forexample, aqueous and non-aqueous sterile suspensions which can includesuspending agents and thickening agents. The formulations can bepresented in unit-dose or multi-dose containers, for example, sealedampules and vials, and can be stored in a lyophilized conditionrequiring, for example, the addition of the sterile liquid carrier,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules and tabletsof the kind previously described.

A pharmaceutically acceptable carrier can additionally contain any of avariety of physiologically acceptable compounds that act, for example,to stabilize the deglutamated compound. Such physiologically acceptablecompounds include, for example, carbohydrates such as glucose, sucroseor dextrans; antioxidants such as ascorbic acid or glutathione;chelating agents such as EDTA, which disrupts microbial membranes;divalent metal ions such as calcium or magnesium; low molecular weightproteins; lipids or liposomes; and other stabilizers or excipients.

A variety of routes of administration can be useful for delivering apharmaceutical composition of the invention. Such routes include thosedescribed previously and will depend, in part, on the particulartherapeutic need and formulation. One skilled in the art understandsthat an effective amount of the deglutamated compound is included in thepharmaceutical composition and depends on the pathological condition tobe treated and the specific physical characteristics of the individualaffected with the disease. Those skilled in the art will know or candetermine a specific regime of administration which is effective for aparticular application using the teachings and guidance provided hereintogether with diagnostic and clinical criteria known within the field ofart of the particular pathological condition.

The invention further provides a kit including the endogenousdeglutamated compound described in Example IV. In one embodiment, thekit further includes methotrexate. Those skilled in the art can readilyincorporate a composition of the invention into kit form in combinationwith appropriate buffers and solutions for the practice of one or moreof the methods disclosed herein.

A kit can include packaging material that houses the contents of thekit. The packaging material is constructed by well known methods,preferably to provide a sterile, contaminant-free environment. Thepackaging materials employed herein in relation to a kit or diagnosticsystem can be any acceptable material that is customarily utilized insmall molecule-based therapeutic or diagnostic systems. A package can beany solid matrix or material such as glass, plastic, paper, foil, andthe like, capable of holding within fixed limits a compound of thepresent invention. Thus, for example, a package can be a glass vial ormicrotiter plate used to contain microgram or milligram quantities ofthe endogenous deglutamated compound disclosed herein in Example IV.

The packaging material can include a label which indicates that thecompositions can be used for reducing toxicity associated withmethotrexate therapy, for determining an intracellular level ofmethotrexate, or for optimizing methotrexate therapy. In addition, thepackaging material can include instructions indicating how the materialswithin the kit are employed. Such instructions will be in accordancewith the description set forth herein for performing the methods. Theinstructions will typically include a tangible expression describing theconcentration of the compound or at least one therapeutic or diagnosticmethod parameter including, without limitation, the relative amounts ofthe compound and methotrexate or some other component to be admixed,maintenance time periods for components of the kit, recommendedtemperature for storage or use, buffer conditions, and the like.

As further disclosed herein, intracellular levels of a deglutamatedcomposition of the invention can be determined and used to calculatedthe intracellular level of methotrexate. Thus, the present inventionprovides a method for determining an intracellular level ofmethotrexate, by deglutamating a cellular extract to produce adeglutamated composition of the invention; and determining the level ofdeglutamated composition, where the level of the deglutamatedcomposition can be correlated with the intracellular level ofmethotrexate. In one embodiment, the cellular extract is a red bloodcellular extract.

The present invention further provides a method of optimizingtherapeutic efficacy or reducing toxicity associated with methotrexatetherapy administered to an individual by deglutamating compounds in acellular extract from the individual; and determining a level of adeglutamated composition of the invention in the cellular extract, wherea drug or dosage subsequently administered to the individual is selectedbased on the level of the deglutamated composition. The level ofmethotrexate can optionally also be determined in the cellular extract;in this case, a drug or dosage subsequently administered to theindividual is selected based on the ratio of the level of methotrexateto the level of the deglutamated composition.

The following examples are intended to illustrate but not limit thepresent invention.

Example I

Quantification of Methotrexate by Fluorimetric Detection ofMethotrexate-Photolytic Products

This example describes quantification of methotrexate using highperformance liquid chromatography (HPLC) and post-column photolysisfollowed by fluorometric detection of photolytic products.

The MTX detection system was based on an HPLC system (Agilent 1100 HPLCchemstation system) equipped with a C18 reversed phase column (25 cm×4.6mm X Terra MS C18, 5 micrometer particle size; Waters, Milford, Mass.),a post-column photochemical reactor unit (Aura Industries; New York,N.Y.) and a post-reactor unit fluorometer detector. The system alsoincluded a C18 pre-column that was changed every 200 injections.

Samples containing 20 to 1000 nmol/L MTX (Sigma, St. Louis, USA) in RBCextracts were run in the HPLC system at a flow rate of 1 ml/min, with a15-minute linear gradient from 2% acetonitrile/98% mobile phase A (10 mMammonium acetate, pH 6.5, unless otherwise indicated and 0.2% hydrogenperoxide in water) to 25% acetonitrile/75% mobile phase A, unlessotherwise indicated. After 15 minutes, mobile phase was returned to 100%mobile phase A and the column was re-equilibrated for 5 minutes. Thesamples were maintained at 4° C. and injected every 20 minutes with anautoinjector.

Eluate from the column passed through the photochemical reactor unit inTEFLON™ tubing ( 1/16 inch outer diameter, 0.25 mm inner diameter, 8meter length) that was connected on-line after the column and before thefluorometer detector and assembled as a knitted coil. The knitted coilwas extended lengthwise through the photochemical reactor unit whichalso housed an elongated 254 nm low pressure mercury ultraviolet lamp(Aura Industries; New York, N.Y.). The time of irradiation of MTX in thephotochemical reactor was 24 seconds. Irradiation for less than 24seconds was achieved by masking all but a portion of the elongated lampwith foil such that only a portion of the knitted coil was irradiated.In particular, the lamp was masked such that only 1 meter of the coilwas irradiated with the lamp which at a flow rate of 1 ml/mincorresponded to 3 seconds irradiation. Methotrexate photolytic productswere measured at an excitation wavelength set at 400 nm and an emissionwavelength at 470 nm, unless otherwise indicated. Chromatograms wereacquired and analyzed on a Hewlett-Packard Vector XP computer. Theretention times described herein for the above-described HPLC systemwere measured from the time of injection to time of detection at thepost-reactor unit fluorometer detector.

As shown in FIG. 2C, HPLC chromatography with in line photolysis underconditions that formed methotrexate photolytic product A, andfluorescence detection at an excitation wavelength of 400 nm andemission wavelength at 464 nm produced a single peak having a retentiontime of about 12.4 minutes. The limit of detection, defined as threetimes the signal-to-noise ratio, was 5 nmol/L for MTX. The analyticalcolumn demonstrated no deterioration of its performance after up to 1000injections.

Two methotrexate photolytic products were obtained from the post-columnderivatization. Product A was formed from the equivalent of a 3-secondirradiation and exhibited an excitation spectrum with a wavelengthmaximum at 274 nm and a smaller peak with a maximum at 380 nm and anemission spectrum with wavelength maximum at 464 nm. FIG. 2D shows thefluorescence excitation and emission spectra for product A of the peakeluting at 12.42 minutes in two separate HPLC separations underconditions similar to those described for the chromatogram of FIG. 2C.Product B was formed following irradiation corresponding to 24 secondsusing an 8 meter knitted reactor coil. Product B had maximum excitationat 370 nm and maximum emission at 417 nm.

The effect of pH on MTX detection was analyzed using the MTX detectionsystem described above except that the mobile phase was isocratic andconsisted of 10 mM ammonium acetate, 0.2% hydrogen peroxide in water,10% acetonitrile and a different pH from 4.0 to 7.0. As shown in FIG.2A, when detection was based on conversion to photolytic product A,increasing pH resulted in an increase in the peak area for MTX. However,the peak area was relatively unchanged at different pH values in thisrange, when detection was based on conversion to photolytic product B.At pH greater than about 4.2, product A exhibited higher fluorescenceintensity compared to product B.

The effect of acetonitrile on MTX detection was analyzed using the MTXdetection system described above except that the mobile phase wasisocratic and consisted of 10 mM ammonium acetate, pH 6.0, 0.2% hydrogenperoxide in water and different percentages of acetonitrile varying from10 to 20%. As shown in FIG. 2B, when detection was based on conversionto photolytic product A, increasing percentages of acetonitrile resultedin a decrease in the peak area for MTX. However, the peak area wasrelatively unchanged at different acetonitrile percentages in thisrange, when detection was based on conversion to photolytic product B.In the range of acetonitrile tested, product A exhibited higherfluorescence intensity compared to product B.

These results indicate that pH and percentage acetonitrile of the mobilephase and the time of irradiation can be changed to modify thesensitivity of MTX detection. In particular, these results indicate thatthe sensitivity of detection of MTX photolytic product A can be changedby altering pH and acetonitrile concentration.

Example II Conversion of Methotrexate Polyglutamates to Methotrexate

This example describes efficient conversion of methotrexatepolyglutamates (MTXPGs) to methotrexate (MTX) and further demonstratesquantitation of methotrexate polyglutamates in a cellular extract.

MTXPGs were enzymatically converted to MTX by incubation with RBCextract and plasma as follows. RBCs were isolated from healthy donors(Blood bank; San Diego, Calif.). Hemolysates were prepared having0.88×10⁹ RBC per 100 μl. For spiked samples, MTXPG (SchirksLaboratories; Jona, Switzerland) was added to RBC extracts at a finalconcentration of 1 μM (from stock solutions containing 100 μM MTXPG in0.1 N potassium hydroxide). Reconstituted plasma was prepared fromlyophilized plasma (Sigma, St. Louis, USA; Cat. No. P9523) and 100 μlwas added to 50 μl of RBC extract, mixed for 30 seconds, and 100 μl ofbuffer containing 100 mM potassium phosphate pH 4.5 and 150 mMmercaptoethanol was added. Because the reconstituted plasma and RBCextract are strongly buffered at physiological pH, the mixture had afinal pH of about 6.5. Samples were incubated for 12-14 hours, unlessotherwise indicated, in the dark at 37° C. After incubation 30 μl of 70%perchloric acid was added to the mixture, vortexed for 15 seconds andcentrifuged for 10 minutes. A total volume of 80 μl was injected ontothe HPLC system for analysis using the MTX detection system described inExample I.

FIG. 3 shows typical chromatograms of an RBC extract blank (Panel A) andan RBC extract spiked with MTXPG₂₋₇ (Panel B, 100 nmol/L RBC) under theabove-described conditions for enzymatic conversion to MTX. Although themaximum excitation wavelength for the methotrexate photolytic productwas 274 nm, excitation wavelength was set at 400 nm to avoid interferingsignals observed in the RBC extract blank. As shown in FIG. 3C, theexcitation spectrum for the MTX photolytic product from the RBC extractthat eluted at 12.5 minutes in the chromatogram shown in FIG. 3B wassimilar to the excitation spectrum for the photolytic product obtainedfrom an MTX standard, measured in water. The overlap in the excitationspectra confirm that the 12.5 minute peak is MTX.

Standard curves demonstrated a linear relationship between peak area andconcentration with correlation coefficients >0.995 for MTX, and for MTXformed after enzymatic conversion of MTXPG₂ (MTXPG having 2 glutamates)or MTXPG₂₋₇ (equimolar mixture of MTXPG₂, MTXPG₃, MTXPG₄, MTXPG₅,MTXPG₆, and MTXPG₇), Spiked standards were assayed in duplicate.Standard curves were fit by linear regression using peak areas versusconcentrations. Equations describing the standard curves were: MTX,y=0.401x+0.281; MTX after enzymatic conversion of MTXPG₂,y=0.383×+0.115; and MTX after enzymatic conversion of MTXPG₂₋₇,y=0.391x−0.122 where y=peak area and x=spiked concentration.

As shown in FIG. 4, MTXPG₂ (Panel A) and MTXPG₂₋₇ (Panel B) werecompletely converted to MTX after an 8 hour incubation at 37° C., in thedark, at concentration of 1000 nmol/L RBC and the conversion was largelycomplete by 4 hours. The percent conversion of MTXPG to MTX wasdetermined by comparing the peak area of MTX formed during the enzymaticconversion at different times with the peak area of MTX spiked in thered blood cells at the same concentration.

The intra-day accuracy and inter-day accuracy for quantitation of MTXand MTXPGs are shown in Table I. RBC extracts were incubated with 20,100 or 500 nmol/L of MTX, MTXPG₂ or MTXPG₂₋₇ (Target concentrations; seeTable I), and the observed concentration of MTX was determined by theHPLC method described in Example I. For the intra-day accuracyassessment, the mean observed MTX concentration was determined for 10spiked replicates at each target concentration (see Table I). The meanobserved MTX concentrations for inter-day accuracy assessment were basedon three replicates from five different days (Table 1). Accuracy wascalculated as the percentage error of the mean observed MTXconcentration from the target concentration value for each set of spikedsamples (mean observed concentration/target concentration×100%).Precision was determined by the relative standard deviation (RSD).

TABLE I Intra-day (n = 10) Inter-day (n = 5) Mean Mean Mean Mean Targetobserved accuracy observed accuracy conc. MTXPG₁ RSD of target MTXPG₁RSD of target Standard nmol/L conc. nmol/L % value (%) conc. nmol/L %value (%) MTXPG₁ 50.0 45.0 4.8 90.0 48.9 8.4 97.9 250.0 250.2 3.8 100.1260.8 3.8 104.3 500.0 504.2 4.1 100.8 517.3 2.3 103.5 MTXPG₂ 50.0 48.21.8 96.4 48.8 6.8 97.7 250.0 231.1 4.2 92.5 238.5 2.7 95.4 500.0 473.23.6 94.6 490.8 4.2 98.2 MTXPG₂₋₇ 50.0 45.4 4.7 90.7 47.0 4.3 94.1 250.0244.5 3.3 97.8 245.6 4.1 98.2 500.0 473.2 2.3 94.6 492.8 6.1 98.6

Highly efficient conversion of MTXPG₂ and MTXPG₂₋₇, respectively, to MTXwas demonstrated by the accuracies of greater than 95% and a coefficientof variation less than 6% for all target concentrations as shown inTable I. Furthermore, as shown in FIG. 5, the plots of observed MTXconcentration versus the concentration of spiked MTXPG₂ or MTXPG₂₋₇ hadslopes of 0.98 and 1.0 respectively, demonstrating that the method wasaccurate and converted greater than 95% methotrexate polyglutamates tomethotrexate. The lower limit of quantification of MTXPG by conversionto MTX and analysis on the MTX detection system was 10 nmol/L MTXPG.

These results demonstrate that MTXPG can be efficiently converted to MTXin RBC extract samples using plasma as a source of gamma glutamylhydrolase. The results further demonstrate that methotrexatepolyglutamates can be accurately and quantitatively detected in RBCextracts by converting the MTXPGs to MTX and quantitating the amount ofMTX by fluorescent detection of a MTX photolytic product.

Example III Monitoring Methotrexate Polyglutamate Levels in PatientsReceiving Low-Dose Methotrexate Therapy

Methotrexate requires intracellular activation to methotrexatepolyglutamate to exert anti-arthritic effects. This example describes amethod for quantitating the levels of intracellular methotrexatepolyglutamates in the RBCs of a patient following administration oflow-dose MTX therapy.

Blood samples (5 ml) from polyarthritis patients receiving low-dose MTXwere collected after written informed consent. The patients had receiveda median of 15 mg MTX (range 2.5 mg to 40 mg per week) for at leastthree months. Blood samples were centrifuged for 10 minutes to separateplasma and buffy coat from RBCs. After washing the RBC pellet with 2volumes of saline, RBC count was determined with a Beckman Coulter OnyxCounter; RBCs were subsequently stored at −70° C. until analysis.Results were normalized to 10 red blood cells. Patient results wereexpressed as average ±SEM (range).

MTXPG concentrations were determined using the methods described inExample II except that RBC extracts were not spiked with MTXPG. MTXPGconcentrations were calculated by subtracting MTX concentrationsmeasured after enzymatic treatment with MTX concentrations measured in acorresponding sample not subject to enzymatic treatment. To quantify MTXconcentrations without enzymatic treatment, a second 100 μl aliquot ofpatient RBC extract was obtained but not treated with plasma or buffer.Rather, 200 μl water was added to the RBC extract prior to immediatedeproteinization with 30 μl 70% perchloric acid as described above.

An exemplary determination of MTXPG concentration in the red blood cellsof a patient on 2.5 mg per week MTX therapy follows. A total of 0.88×10⁹RBC cells were obtained from the patient and analyzed and resulted inthe chromatogram shown in FIG. 6A. The fluorescence excitation spectrumof photolytic product A produced from the peak eluting at 12.42 minutesin the chromatogram of FIG. 6A was similar to the excitation spectrumfor methotrexate's photolytic product in water as shown in FIG. 6B. Theoverlap in the excitation spectra confirm that the 12.42 minute peak isMTX.

The concentration of MTXPG in the sample was determined to be 7.9 pmolMTXPG/10⁹ cells by subtracting the MTX concentration prior to enzymaticconversion, which was 16.5 pmol MTXPG/10⁹ cells, from MTX afterenzymatic conversion, which was 28.6 pmol MTXPG/10⁹ cells. Based onthese results, it was determined that 27% of the total MTX in thepatient's RBCs was present as a polyglutaminated derivative.

In 12 patients, total MTXPG ranged from 3.4 to 37.0 pmol MTXPG/10⁹ cellswith a median of 16.5 pmol MTXPG/10⁹ cells. Prior to conversion, MTXconcentration ranged from 1.9 to 30.0 pmol/10⁹ cells. The percentage ofMTXPG in the patients' RBCs ranged from 35% to 100% of total MTX with amedian of 45%.

These results demonstrate an assay that is capable of detecting theamount of RBC intracellular MTX in a patient on low dose MTX therapy aswell as the amount of this MTX that has been converted to the activeMTXPG form.

Example IV A Compound Isolated from a Deglutamated Red Blood CellularExtract

This example describes isolation and characterization of a compoundisolated from red blood cells following deglutamation.

RBCs were isolated, hemolysates prepared and the hemolysatesdeglutamated by incubation with reconstituted plasma as described inExample II. Deglutamated samples were injected onto the HPLC system foranalysis using the MTX detection system described in Example I.

As shown in FIG. 3A, an endogenous peak having a retention time of 11.90minutes was detected in the deglutamated RBC extract. The endogenouspeak had a retention time of 11.91 minutes in a deglutamated RBC extractthat had been spiked with MXTPG and was resolved from the MTX peak,which had a retention time of 12.5 minutes (FIG. 3B). In a deglutamatedRBC extract from a patient undergoing methotrexate therapy, theendogenous peak had a retention time of 12.02 minutes and was againresolved from the MTX peak, which had a retention time of 12.42 minutes(FIG. 6). In both the MTXPG spiked sample and patient sample, theresolution was sufficient to allow the endogenous peak to be integratedfor quantitative comparison between samples. The endogenous peak hadretention times that were about 0.6 to 0.4 minutes less than theretention times for MTX.

A fluorescence excitation spectrum was obtained for the material elutingwith the endogenous peak by detecting emission at 464 nm for the HPLCcolumn eluate having a retention time of 12.02 minutes. As shown in FIG.7, the excitation spectrum had peak maxima at about 299 nm and 402 nmand minima at about 259 nm, 336 nm and 429 nm.

The excitation spectrum of the endogenous peak shown in FIG. 7 wasobtained following 3 second photolysis under the conditions set forth inExample I. Also shown in FIG. 7 is the excitation spectrum obtained forthe endogenous peak under similar HPLC conditions except that the samplewas not irradiated prior to detection. The spectra for the endogenouspeak with and without photolysis were similar, indicating that theendogenous compound isolated from deglutamated red blood cellularextracts is photostable to UV irradiation for at least 3 seconds in thepresence of peroxide.

The endogenous compound that is present in the endogenous peak isobtained by isolating from the column eluate the fraction correspondingin retention time to the endogenous peak. The presence of the compoundin the isolated fraction is confirmed by analysis of a portion of thefraction. This portion is analyzed by reinjection onto the HPLC systemusing the post-column photolysis and fluorescence detection systemdescribed in Example I and the presence of the compound identified byelution at the appropriate time.

The endogenous compound is characterized by co-injection with knownfolates in the HPLC system or other analytical chromatography system. Aknown folate that coelutes with the endogenous compound is identified aspotentially having similar structure. Further characterization of theendogenous compound is based on the rate at which it is produced by GGHcompared to the rate at which a known polyglutamated folate is convertedto a monoglutamated folate or the photostability of the endogenouscompound compared to photostability of a known folate. Similarity in thereaction rates, photostability or both also identifies the endogenouscompound as having a structure that is similar to the known folate.

The molecular weight of the endogenous compound is determined by massspectrometry using methods known in the art. Further structuralcharacterization is carried out by nuclear magnetic resonancespectroscopy using methods known in the art as applied to analysis ofmethotrexate and other folates.

Throughout this application various patent and non-patent publicationshave been referenced. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

The term “comprising” is intended herein to be open-ended, including notonly the recited elements, but further encompassing any additionalelements.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the claims.

1. A method for predicting therapeutic efficacy or reducing toxicityassociated with methotrexate (MTX) therapy administered to anindividual, comprising the steps of: (a) converting methotrexatepolyglutamates (MTXPGs) to MTX in a cellular extract from saidindividual under conditions suitable for efficient conversion of MTXPGsto MTX; (b) determining a level of MTX in said cellular extract, saidlevel of MTX correlating with a level of MTXPGs, thereby determining alevel of MTXPGs in said cellular extract, wherein a drug or dosagesubsequently administered to said individual is selected based on saidlevel of MTXPGs.